CN107828879A - A kind of PKP2 gene mutation detection kits and its detection method - Google Patents
A kind of PKP2 gene mutation detection kits and its detection method Download PDFInfo
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- CN107828879A CN107828879A CN201710975176.2A CN201710975176A CN107828879A CN 107828879 A CN107828879 A CN 107828879A CN 201710975176 A CN201710975176 A CN 201710975176A CN 107828879 A CN107828879 A CN 107828879A
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- pkp2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a kind of PKP2 gene mutation detection kits, include the primer pair of R158K, Q211X, L419S and N852fsX930 pleomorphism site on specific amplification PKP2 genes, the primer pair includes common upstream primer and parting anti-sense primer, and the parting anti-sense primer includes wild type special primer and saltant type special primer.The detection method of above-mentioned PKP2 gene mutation detection kits includes:Amplification uses PCR while to 4 pleomorphism sites of the PKP2 genes in sample, and the detection of its amplified production uses high performance liquid chromatography, multi-fluorescence Capillary Electrophoresis or polyacrylamide gel electrophoresis.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of PKP2 gene mutation detection kits.
Background technology
Arrhythmogenic right ventricular cardiomyopathy (arrhythmogenic right ventricular
Cardiomyopathy/dysplasia, abbreviation ARVC/D) be it is a kind of with fiber, adipose tissue progressive substitution cardiac muscular tissue
Autosomal dominant inherited disease, with incomplete penetrance, thus cause right ventricle expansion and the ventricular rhythm originating from right ventricle
It is not normal, it can behave as palpitaition, syncope, heart failure or even die suddenly, one of the main reason for being teenager's sudden cardiac death, especially
It is Exercise-related sudden death.
It is now recognized that ARVC/D is a kind of heredity desmosome cardiomyopathies.By a series of pathophysiological change, finally
Cause clinical arrhythmia cordis, heart failure and sudden death.The PKP2 albumen of PKP2 gene codes be desmosome albumen important composition into
Point, the change of its structure influences whether the function of desmosome.PKP2 gene introns and exons mutation are possible to influence PKP2
The structure of albumen.
At present, clinically mainly there are imageological examination and laboratory inspection for the conventional diagnostic techniques of genetic cardiomyopathies
Look into two major classes.Color Sonography, heart CT and heart nuclear magnetic resonance are generally comprised with regard to imageological examination.Although these can be more accurate
Ground judges the objective indicator such as ventricular wall thickness, chambers of the heart size, but also can only be roughly to judge whether patient suffers from the heredity heart
Myopathy, and can not accurately, specificity definite judgement is made to specific species of genetic cardiomyopathies etc..Laboratory examination, mainly
The detection of conventional biochemical index, such as cardiac troponin, atrial natriuretic peptide are included, is only capable of reflecting heart injury, it is impossible to losing
The particular type and its gene mutation situation of transmissibility cardiomyopathy make a distinction.Such as examined using the method adjuvant clinical of molecular biology
Disconnected, that can substantially reduces the compliance that is damaging, improving patient of patient.
Meanwhile traditional gene tester based on Sanger sequencings has the drawbacks of flux is low, primary first-order equation can only
An amplification region is detected, can not meet that huge genetic test region and multisample detect ageing requirement.Therefore, having must
Seek a kind of method of new detection Arrhythmogenic right ventricular cardiomyopathy (ARVC/D) gene mutation, it is accurate to improve diagnosis
Property, reduce cost and labor intensity and improve ageing
The content of the invention
The present invention provides a kind of high accuracy, low cost, high flux, quick detection kit, available for detecting and cause
The mutation type of the related PKP2 genes of arrhythmia cordis right ventricular cardiomyopathy (ARVC/D).
A kind of PKP2 gene mutation detection kits, including on specific amplification PKP2 genes R158K, Q211X,
The primer pair of L419S and N852fsX930 pleomorphism sites, the primer pair include common upstream primer and parting anti-sense primer,
The parting anti-sense primer includes wild type special primer and saltant type special primer, corresponding 4 pleomorphism sites it is public on
Primer, wild type special primer and saltant type specific primer sequences are swum as shown in SEQ ID № 1~12.
Wherein, the 5' ends of common upstream primer of the sequence as shown in SEQ ID № 1,4,7,10 are marked with fluorescence labeling dye
Material, the fluorescent dye are selected from FAM, HEX, TAMRA, ROX, Cy3, Cy5, JOE, VIC, PET.
The PKP2 gene mutation detection kits also include gene order contrast template, the gene order contrast template
Including normal sequence template SEQ ID № 13~14, and mutant nucleotide sequence template SEQ ID № 15~16.
The PKP2 gene mutation detection kits also include PCR buffer solutions, hot start Taq polymerase, ultra-pure water, fluorescence molecule
Measure internal standard and allelic ladder.
The detection method of above-mentioned PKP2 gene mutation detection kits includes:It is polymorphic to 4 of the PKP2 genes in sample
Property site amplification use PCR, the detection of its amplified production is using high performance liquid chromatography, multi-fluorescence capillary
Electrophoresis tube or polyacrylamide gel electrophoresis.
Further, the amplification program of the PCR is:95 DEG C 2 minutes;94 DEG C 30 seconds, 59 DEG C 30 seconds, 72
DEG C 30 seconds, totally 10 circulations;94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, totally 18 circulation;72 DEG C 10 minutes.
The beneficial effects of the invention are as follows:
(1) based on PCR with improving pleomorphism site detection sensitivity, false positive is substantially reduced;
(2) common upstream primer can at most detect 8 not simultaneously by being equipped with different fluorophors under primary first-order equation
Same sample, has the characteristics of efficient economizing;
(3) PCR is completed within 1.5 hours, capillary electrophoresis detection process also less than 1 hour, whole
Individual detection process was completed in 3 hours, had the characteristics of used time is few, and the quick and degree of accuracy is high;
(4) the directly hands-free DS of sample, DNA extraction steps can be saved, saliva card, FTA cards and blood filter paper can be direct
Amplification, reduces operating procedure, simplifies its operating process;
(5) present invention can accurate judgement be normal and the Genotyping of sudden change sample.
Brief description of the drawings
Fig. 1 is the detection collection of illustrative plates of embodiment 1;
Fig. 2-5 is the detection collection of illustrative plates of embodiment 2 (in each accompanying drawing:A is normal parting schematic diagram, and B is mutation parting signal
Figure);
Fig. 6 is the detection collection of illustrative plates of embodiment 3;
Fig. 7 is the detection collection of illustrative plates of embodiment 4;
Fig. 8 is the detection collection of illustrative plates of embodiment 5;
Fig. 9 is the detection collection of illustrative plates of embodiment 6.
Embodiment
The present invention is described in further detail below by embodiment combination accompanying drawing.But those skilled in the art
It will be understood that the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted in embodiment
Particular technique or condition person, according to the technology described by document in the art or condition (such as with reference to J. Pehanorm Brookers etc.
Write, what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.Institute
With reagent or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.
PKP2 gene mutation detection kits, including R158K, Q211X, L419S on specific amplification PKP2 genes
With the primer pair of N852fsX930 pleomorphism sites, the primer pair includes common upstream primer and parting anti-sense primer, described
Parting anti-sense primer includes wild type special primer and saltant type special primer, and the common upstream of corresponding 4 pleomorphism sites is drawn
Thing, wild type special primer and saltant type specific primer sequences are as shown in SEQ ID № 1~12.Wherein, sequence such as SEQ ID
Shown in № 1 for R158K pleomorphism site common upstream primers, sequence as shown in SEQ ID № 2 for R158K polymorphisms
Site wild type special primer, sequence as shown in SEQ ID № 3 for R158K pleomorphism site saltant type special primers;Sequence
Arrange and be as shown in SEQ ID № 5 for Q211X pleomorphism site common upstream primers, sequence as shown in SEQ ID № 4
Q211X pleomorphism site wild type special primers, sequence as shown in SEQ ID № 6 for Q211X pleomorphism site saltant types
Special primer;Sequence as shown in SEQ ID № 7 for L419S pleomorphism site common upstream primers, sequence such as SEQ ID №
Shown in 8 for L419S pleomorphism site wild type special primers, sequence as shown in SEQ ID № 9 for L419S polymorphic positions
Point mutation type special primer;Sequence as shown in SEQ ID № 10 for N852fsX930 pleomorphism site common upstream primers,
Sequence as shown in SEQ ID № 11 for N852fsX930 pleomorphism site wild type special primers, sequence such as SEQ ID №
Shown in 12 for N852fsX930 pleomorphism site saltant type special primers.Public affairs of the sequence as shown in SEQ ID № 1,4,7,10
The 5' ends of sense primer are marked with fluorescent marker dyes altogether, the fluorescent dye be selected from FAM, HEX, TAMRA, ROX, Cy3, Cy5,
JOE、VIC、PET.The PKP2 gene mutation detection kits also include gene order contrast template, the gene order control
Template includes normal sequence template SEQ ID № 13~14, and mutant nucleotide sequence template SEQ ID № 15~16.It is described
PKP2 gene mutation detection kits also include PCR buffer solutions, hot start Taq polymerase, ultra-pure water, fluorescence molecule amount internal standard and equipotential
Genotype standard substance.
It is specially as shown in table 1:
The composition of table 1, kit
The specific detection method of kit of the present invention:
1st, the extraction of sample DNA
According to the actual conditions of sample, suitable method extraction sample DNA is selected, common method has:Paramagnetic particle method, it is cloudy from
Sub-exchange resin method and alkaline lysis etc..If sample is blood filter paper, FTA cards, saliva card etc., step can be simplified, directly expanded.
2nd, PCR reacts
The amplification system of PCR reactions is as follows:
The amplification program of PCR reactions is as follows:
3rd, genetic analyzer fluorescence electrophoresis detect
12 μ L deionized formamides mix with 0.5 μ L molecular weight internal standard (AGCU Marker SIZ-500).Add 1 μ L
PCR reaction products or PKP2Allelic Ladder, it is vortexed and mixes 3500r/min centrifugations 2min, 95 DEG C are denatured 3min, ice bath
3min, tested and analyzed with genetic analyzer ABI3130XL.
Embodiment 1, the foundation of standard diagram:
By above-mentioned detection method, crt gene sequence is detected, and obtains normal sequence control wild type as shown in Figure 1
4 site primer standard partings of 4 site primer collection of illustrative plates and mutant nucleotide sequence the control saltant type special primer of special primer
Collection of illustrative plates.
Embodiment 2, by above-mentioned detection method, using PKP2 gene detecting kits detect respectively R158K, Q211X,
The mono- detection sites of L419S and N852fsX930, the parting using normal or mutant nucleotide sequence as template contrasts, as a result such as the institute of Fig. 2~5
Show.
Embodiment 3, No.1 sample detection:
No.1 sample is detected by above-mentioned application method, obtains collection of illustrative plates as shown in Figure 6, it is known that No.1 sample is
L491S and N852fsX930 sites double-site mutant.
Embodiment 4, No. two sample detections:
No. two samples are detected by above-mentioned application method, obtain collection of illustrative plates as shown in Figure 7, it is known that No. two samples are
The site mutation of L491S, R158K and N852fsX930 site three.
Embodiment 5, No. three sample detections:
No. three samples are detected by above-mentioned application method, obtain collection of illustrative plates as shown in Figure 8, it is known that No. three samples are
N852fsX930 single-site mutants.
Embodiment 6, No. four sample detections:
No. four samples are detected by above-mentioned application method, obtain collection of illustrative plates as shown in Figure 9, it is known that No. four samples is just
Often do not undergo mutation.
SEQUENCE LISTING
<110>The magnificent Zhong Yuan bio tech ltd in Guangdong
<120>A kind of PKP2 gene mutation detection kits and its detection method
<130> 2017
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence
<400> 1
gggaagagga acagcacagt aca 23
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
gctgtgcgtg tagtgagccc 20
<210> 3
<211> 16
<212> DNA
<213>Artificial sequence
<400> 3
tgcgtgtagt gagcct 16
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<400> 4
gggaagagga acagcacagt aca 23
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence
<400> 5
atgtgtcaaa gtggcgctg 19
<210> 6
<211> 23
<212> DNA
<213>Artificial sequence
<400> 6
tggtatgtgt caaagtggcg cta 23
<210> 7
<211> 22
<212> DNA
<213>Artificial sequence
<400> 7
caggttaagc agcttcgtgg ca 22
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence
<400> 8
ctaagtttct caaggcccca 20
<210> 9
<211> 24
<212> DNA
<213>Artificial sequence
<400> 9
aatactaagt ttctcaaggc cccg 24
<210> 10
<211> 22
<212> DNA
<213>Artificial sequence
<400> 10
catccagaag cctcactcat tc 22
<210> 11
<211> 24
<212> DNA
<213>Artificial sequence
<400> 11
cttcttgtag gcatgatgca gttc 24
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
cttgtaggca tgatgcagtt 20
<210> 13
<211> 309
<212> DNA
<213>Artificial sequence
<400> 13
gctggggaag aggaacagca cagtacagct cccagaagtc cgtggaagaa aggtccttga 60
ggcatcctct gaggagactg gagatttctc ctgacagcag cccggagagg gctcactaca 120
cgcacagcga ttaccagtac agccagagaa gccaggctgg gcacaccctg caccaccaag 180
aaagcaggcg ggccgccctc ctagtgccac cgagatatgc tcgttccgag atcgtggggg 240
tcagccgtgc tggcaccaca agcaggcagc gccactttga cacataccac agacagtacc 300
agcatggct 309
<210> 14
<211> 397
<212> DNA
<213>Artificial sequence
<400> 14
agcagttgag gagcgaagag gtcttacaga acacccacag gccgcatcca gaagcctcac 60
tcattctccc tgatctcaga atgtcctttt cttttcagct atgcctccaa caaagcaagt 120
aaagctgctt ccgtccttct gtattctctg tgggcacaca cgaactgcat catgcctaca 180
agaaggtaag aacatgaaca aggaatgtgt catagttaac agagccaggt aataatggcc 240
cacttgactt gaccctgata tgcatctgct tcttccccag gttaaccagc ttcgtggcat 300
cctcaagctt ctgcagctcc taaaagttca gaatgaagac gttcagcgag ctgtgtgtgg 360
ggccttgaga aacttagtat ttgaagacaa tgacaac 397
<210> 15
<211> 309
<212> DNA
<213>Artificial sequence
<400> 15
gctggggaag aggaacagca cagtacagct cccagaagtc cgtggaagaa aggtccttga 60
ggcatcctct gaggagactg gagatttctc ctgacagcag cccggagaag gctcactaca 120
cgcacagcga ttaccagtac agccagagaa gccaggctgg gcacaccctg caccaccaag 180
aaagcaggcg ggccgccctc ctagtgccac cgagatatgc tcgttccgag atcgtggggg 240
tcagccgtgc tggcaccaca agcaggtagc gccactttga cacataccac agacagtacc 300
agcatggct 309
<210> 16
<211> 225
<212> DNA
<213>Artificial sequence
<400> 16
tgcctacaag aaggtaagaa catgaacaag gaatgtgtca tagttaacag agccaggtaa 60
taatggccca cttgacttga ccctgatatg catctgcttc ttccccaggt taaccagctt 120
cgtggcatcc tcaagcttct gcagctccta aaagttcaga atgaagacgt tcagcgagct 180
gtgtgcgggg ccttgagaaa cttagtattt gaagacaatg acaac 225
Claims (7)
1. a kind of PKP2 gene mutation detection kits, it is characterised in that including for specific amplification PKP2 genes
The primer pair of R158K, Q211X, L419S and N852fsX930 pleomorphism site, the primer pair include common upstream primer and
Parting anti-sense primer, the parting anti-sense primer include wild type special primer and saltant type special primer, corresponding 4 polymorphisms
Common upstream primer, wild type special primer and the saltant type specific primer sequences in site are as shown in SEQ ID № 1~12.
2. PKP2 gene mutation detection kits according to claim 1, it is characterised in that the 5' of each common upstream primer
End is marked with fluorescent marker dyes.
3. PKP2 gene mutation detection kits according to claim 2, it is characterised in that the fluorescent dye is selected from
FAM、HEX、TAMRA、ROX、Cy3、Cy5、JOE、VIC、PET。
4. PKP2 gene mutation detection kits according to claim 1, it is characterised in that also compareed including gene order
Template, the gene order contrast template include normal sequence template SEQ ID № 13~14, and mutant nucleotide sequence template SEQ
ID № 15~16.
5. PKP2 gene mutation detection kits according to claim 1, it is characterised in that also including PCR buffer solutions, heat
Start Taq enzyme, ultra-pure water, fluorescence molecule amount internal standard and allelic ladder.
6. a kind of detection method of PKP2 gene mutation detection kits, it is characterised in that using as described in Claims 1 to 5
Amplification of the PKP2 gene mutation detection kits to 4 pleomorphism sites of the PKP2 genes in sample use polymerase chain
Reaction, the detection of its amplified production use high performance liquid chromatography, multi-fluorescence Capillary Electrophoresis or polyacrylamide gel electrophoresis.
7. the detection method of PKP2 gene mutation detection kits according to claim 6, it is characterised in that polymerase chain
Formula reaction amplification program be:95 DEG C 2 minutes;94 DEG C 30 seconds, 59 DEG C 30 seconds, 72 DEG C 30 seconds, totally 10 circulation;94 DEG C 30 seconds,
58 DEG C 30 seconds, 72 DEG C 30 seconds, totally 18 circulation;72 DEG C 10 minutes.
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013013206A1 (en) * | 2011-07-21 | 2013-01-24 | The Board Of Trustees Of The Leland Stanford Junior University | Cardiomyocytes from induced pluripotent stem cells from patients and methods of use |
CN104232773A (en) * | 2014-06-11 | 2014-12-24 | 国家体育总局体育科学研究所 | SNP (single nucleotide polymorphism) compound detection system for screening exercise-induced cardiac accidents |
CN106399487A (en) * | 2016-08-31 | 2017-02-15 | 广东华美众源生物科技有限公司 | Method and kit for detecting human fructosediphosphate aldolase B gene |
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2017
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2013013206A1 (en) * | 2011-07-21 | 2013-01-24 | The Board Of Trustees Of The Leland Stanford Junior University | Cardiomyocytes from induced pluripotent stem cells from patients and methods of use |
CN104232773A (en) * | 2014-06-11 | 2014-12-24 | 国家体育总局体育科学研究所 | SNP (single nucleotide polymorphism) compound detection system for screening exercise-induced cardiac accidents |
CN106399487A (en) * | 2016-08-31 | 2017-02-15 | 广东华美众源生物科技有限公司 | Method and kit for detecting human fructosediphosphate aldolase B gene |
Non-Patent Citations (3)
Title |
---|
KEIKO SONODA等: "Quantitative analysis of PKP2 and neighbouring genes in a patient with arrhythmogenic right ventricular cardiomyopathy caused by heterozygous PKP2 deletion", 《EUROPACE》 * |
XIAOLIANG QIU等: "Mutations of plakophilin-2 in Chinese with arrhythmogenic right ventricular dysplasia/cardiomyopathy", 《THE AMERICAN JOURNAL OF CARDIOLOGY》 * |
刘欣等: "致心律失常性右心室心肌病患者PKP2基因突变检测及临床分析", 《中华内科杂志》 * |
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